Several sensor technology developments have been described in the art. In particular, arrays of semiconductor sensors having sensitive and chemically-diverse interface materials capable of interacting with analytes of complex mixtures have previously been used. These can incorporate many operating principles including: doped tin-oxide gas sensors, doped conductive polymers, field effect transistor (FET) devices, and optical fiber devices.
Some sensors are based on a more specific chemical adsorption. For example, enzymes or antibodies can provide a more selective response when incorporated with sensors such as immunoFET's, redox enzyme electrodes, ion-channel simulating devices or antibody-coated piezoelectric or surface acoustic wave devices [review by Gardner J. W., Bartlett P. N., Sensors Actuators, B18–19, 211–220, 1994]. Recently, mass sensing quadrupole transducers (e.g., using mass spectrometers (MS)) have been commercially available for the application in food quality control. MS can analyze sample headspace [U.S. Pat. No. 5,363,707 to Augenblick, et al.].
Generally, multisensor arrays do not include only biosensors. All the wells of a 96 well plate can be viewed concurrently with a charge coupled device (CCD) camera. For example, see Atwood in U.S. Pat. No. 5,766,889. One example of DNA detection by arrays of biosensors uses a fiber optic bundle to screen or analyze a surface enhanced Raman scattering (SERS) surface [U.S. Pat. No. 5,814,516 to Vo-Dinh, Tuan]. Another example of biosensor arrays uses a fiber optic bundle modified with compounds that bind biological species [U.S. Pat. No. 5,837,196 to Pinkel, Daniel]. These examples do not use multivariate processing. Furthermore, these examples have large complex fiber optic bundles. Better MSAs (though not necessarily optical) have smaller and easier to replace sensors. This is important due to fouling of the sensors surfaces.
Other work and examples of devices used in the art of chemical sensing are described into the following references, which illustrate some of the major transducing systems such as (i) resonant microstructures [U.S. Pat. No. 5,025,346 to Tang, Williams C. and Howe, Roger T.; U.S. Pat. No. 5,445,008 to Wachter, Eric A. and Thundat (using a micro-cantilever), Thomas G.; Barnes J. R., et al., Rev. Sci. Instrum., 65(12), 3793–98, 1994], (ii) amperometric, conductivity/capacitance sensing platforms [U.S. Pat. No. 5,801,297 to Mifsud J. -C. and Moy L.] or (iii) optical detection [U.S. Pat. No. 5,563,707 to Prass Werner, et al.,; U.S. Pat. No. 5,512,490 to Walt David R. and Kauer John S.; U.S. Pat. No. 5,004,914 to Vali, Victor, et al.; U.S. Pat. No. 5,436,167 to Robillard Jean J.; U.S. Pat. No. 5,015,843, Seitz, William R. and McCurley, Marian F.].
In particular, the last approach can be illustrated by the development efforts of Walt, et al. [Dickinson T. A., White J., Kauer J. S., Walt D. R., Nature, 382, 697–700, Aug. 22, 1996] employing a miniaturized array of fibers containing a special fluorescent dye (Nile Red) embedded in a polymer matrix. Such a strategy is based on the use of a dye exhibiting large wavelength shifts in its strong fluorescent response to various vapors. The dye is photochemically stable and can be immobilized in polymers. The art uses a configuration of nineteen 300 μm optical fibers with their flat sensing ends coated with the dye (i.e., Nile Red) encapsulated in various polymers. A video frame grabber permits fluorescent intensity versus time data to be recorded. From the variations in response time and the individual fiber light output, the system can be trained to recognize these differences and identify specific vapors. One particular problem with the above-described system involves the photobleaching of the dye molecules. This limits the reliability and reproducibility of the sensor system.
Efforts to date in the art of gas sensor technology have centered upon the field of applications of the detection of toxic substances and smells for the evaluation of odor properties in consumer products, environmental science, and medicine [U.S. Pat. Nos. 5,801,297 and 5,563,707]. For example, microorganism detection in the manner described by Payne, et al. in U.S. Pat. No. 5,807,701, uses an array of conducting polymer sensors to sample vapor associated with the microorganisms. Payne discloses detection of organisms, but not of biomolecules and/or PCR products.
Almost all analytical techniques or process monitoring techniques are based on one or two variables at a time. For example, these variables can be an analytical signal representing absorbance, chromatographic retention time, or electrochemical response. These variables can be time-based as in process control. These traditional analytical techniques usually give acceptable results when analyzing simple systems with only a few components with variables (e.g., analytical signals) that do not interact with each other. Multicomponent mixtures such as solutions, gases, solids, process streams, effluents, and contents of reaction chambers usually have many variables that interact with each other. For example, absorbance peaks of several components can overlap each other. These variables should be analyzed simultaneously to optimize the useful analytical data that would be obscure in traditional analytical techniques.
As explained below, multicomponent mixtures or solutions can be analyzed by a multivariate analysis based on the reference data. {H. M. Heise, A. Biftner, “Multivariate calibration for near-infrared spectroscopic assays of blood substrates in human plasma based on variable selection using partial least squares (PLS) regression vector choices, Fresenius' Journal of Analytical Chemistry, 362(1) (1998) 141–147}. Nonlinear multivariate calibration methods have been reviewed by Sekulic, et al. (Analytical Chemistry, 65 (1993) 835A–845A).
Multivariate analysis techniques include many artificial intelligence techniques. Some examples include artificial neural networks (ANN), expert systems (ESs), fuzzy logic (FL), genetic algorithms (GAs). ANNs learn by training. ESs are based on defined rules. FL systems are based on uncertainty and partial truths. These techniques can be used in concert. New techniques will also be developed, and are contemplated to be used in this invention.
Isidore Rigoutsos and Andrea Califano in U.S. Pat. No. 5,752,019 and related references describe a family of new techniques using probabilistic indexing algorithms such as Fast Look-up Algorithm for String Homology (FLASH), hash algorithms, and data mining algorithms. Paul Stolorz, et a/. describe Bayes algorithms (also called Bayesian statistical methods) in “Predicting Protein Secondary Structure Using Neural Net and Statistical Methods” J. Mol. Biol. 225 (1992) 363.
Multivariate analysis reconstructs analytical data from several variables. Multivariate process monitoring handles noise and drift better with fewer false alarms than univariate monitoring. For example, multivariate analysis can determine an analyte's concentration from several of its absorbance peaks. Theoretically, multivariate analysis can be applied to most analytical and process control techniques. Examples include: fluorescence, chromatography, absorption spectroscopy, emission spectroscopy, X-ray methods, radiochemical methods, nuclear magnetic resonance spectroscopy, electron spin resonance spectroscopy, surface science techniques, refractometry, interferometry, mass spectrometry, gas density, magnetic susceptibility, electrochemistry, surface acoustic wave sensors, sensor arrays, ultrasonic sensors, and thermal analysis.
Multiple variables can be converted into useful analytical data by multivariate analysis. This multivariate analysis or multivariate technique can relate instrumental response to the concentrations, physical, chemical, or physico-chemical properties at several wavelengths. The most commonly used multivariate techniques with FTIR spectroscopy are classical least squares (CLS) and inverse least squares (ILS). CLS is a limited method in the sense that the concentrations of all component analytes must be known exactly. ILS is a wavelength limited method because the number of wavelengths used must be smaller than the number of samples. These two methods lack the power to handle data with similar spectral features. {D. Qin and P. R. Griffiths, SPIE, 2089, p. 548 (1994)}. There are also the factor-based or bilinear projection methods of PLS, sometimes called Projection to Latent Structures, Principal Components Analysis (PCA), and Principle Components Regression (PCR). PLS is a good technique for process control when both process and product data are used to control the process. {Stone, M. Brooks, R. J. (1990) “Continuum Regression: Cross-validated Sequentially Constructed Prediction Embracing Ordinary Least Squares, Partial Least Squares and Principal Components Regression”, Journal of the Royal Statistical Society B, 52, 237–269}. Nonlinear Principle Components Regression (NLPCR) and Nonlinear Partial Least Squares (NLPLS) can model nonlinear responses. {Sekulic, et al., Analytical Chemistry, 65 (1993) 835A–845A}. Soft Independent Modeling of Class Analogy (SIMCA) is another of several more multivariate methods.
The results of the multivariate analysis are usually used directly to give concentration values for the measured analyte. Multivariate techniques can be used for infrared and near-infrared spectroscopy. See James M. Brown, U.S. Pat. No. 5,121,337; Bruce N. Perry, et al., U.S. Pat. No. 5,641,962. Perry, et al. claim non-linear multivariate methods. Multivariate techniques can be used for hyphenated chromatography like GC-MS. Ashe, et al., U.S. Pat. No. 5,699,270. Multivariate techniques can be used for surface acoustic wave (SAW) vapor sensors. Lokshin, et al. U.S. Pat. No. 5,465,608. Some references describe applying other properties to the data such as octane values. Bruce N. Perry, et al., U.S. Pat. No. 5,641,962; Maggard, U.S. Pat. No. 5,349,189, freezing point depression in milk, Arnvidarson et al., U.S. Pat. No. 5,739,034, and cancerous stages of tissue samples Haaland et al. U.S. Pat. No. 5,596,992. There is little art on fitting the data from multivariate analysis to an algorithm to determine other properties. For example, there is little art on joining multivariate analysis with monitoring of any PCR process.
DNA and similar large biomolecules are hard to detect in gas phase. These large biomolecules are hard to volatilize and are subject to degradation. There are only a few methods that can safely break up large biomolecules into detectable fragments such as fast atom bombardment (FAB) and Cf-252 mass spectrometry. For example, see “Fragmentation of Proteins in the 13–29 kDa Mass Range Observed by 252Cf-Plasma Desorption Mass Spectrometry” D. M. Bunk, and R. D. Macfarlane. Proc. Nat. Acad. Sci. (USA) 89 (1992) 6215. Other methods use thermospray or electrospray. For example see Kaufman et al. in “Analysis of Biomolecules using Electrospray”, J. Aerosol Sci., 29, p. 537 (1998). and by Jarell, J. A. and Tomany, M. J. in U.S. Pat. No. 5,828,062. Combining these methods would greatly benefit the detection of large biomolecules such as DNA and RNA.
The Polymerase Chain Reaction (PCR) technique was devised by Kary Mullis in the mid-1980s and, like DNA sequencing, has revolutionized molecular genetics by making possible a whole new approach to the study and analysis of genes. A major problem in analyzing genes is that they are rare targets in a complex genome that in mammals may contain as many as 100,000 genes. Molecular genetics techniques currently used to overcome this problem are very time-consuming, involving cloning and methods for detecting specific DNA sequences. The Polymerase Chain Reaction has changed all this by enabling production of enormous numbers of copies of a specified DNA sequence without resorting to cloning.
The Polymerase Chain Reaction (PCR) exploits certain features of DNA replication. DNA polymerase uses single-stranded DNA as a template for the synthesis of a complementary new strand. These single-stranded DNA templates can be produced by simply heating double-stranded DNA to temperatures near boiling. DNA polymerase also requires a small section of double-stranded DNA to initiate (“prime”) synthesis. Therefore the starting point for DNA synthesis can be specified by supplying an oligonucleotide primer that anneals to the template at that point. In this important feature of the PCR, DNA polymerase can be directed to synthesize a specific region of DNA.
Both DNA strands can serve as templates for synthesis, provided an oligonucleotide primer is supplied for each strand. For a PCR, the primers are chosen to flank the region of DNA that is to be amplified so that the newly synthesized strands of DNA, starting at each primer, extend beyond the position of the primer on the opposite strand. Therefore new primer binding sites are generated on each newly synthesized DNA strand. The reaction mixture is again heated to separate the original and newly synthesized strands, which are then available for further cycles of primer hybridization, DNA synthesis, and strand separation. The net result of a PCR is that the end of n cycles, the reaction contains a theoretical maximum of 2n double-stranded DNA molecules that are copies of the DNA sequence between the primers. This second important feature of PCR results in the “amplification” of the specified region.
A number of amplification reaction amplification reaction assays have been developed, such as the Polymerase Chain Reaction (PCR) described above and in the following references [N. Amheim, H. Erlich, Ann. Rev. Biochem., 1992, 61, pp. 131–56; U.S. Pat. Nos. 4,683,202; 4,683,195; and U.S. Pat. No. 4,985,188 to Mullis, Kary]. In most commonly used sequencing methods, a target-specific sequence is amplified by enzyme cycle reactions ultimately effecting an increase in the amount of amplification unit or amplicon produced. In addition to the target sequence of a typical amplification, additional components and/or reagents can be consumed limiting the amplification of PCR products. Several improvements of the standard PCR method have been reported into literature for various specific applications, such as in-situ, reverse transcriptase, hot start, long and accurate or even touch down methods [Dan R. H., Cox P. T., Wainwright B. J., Baker K., Mattick J. S., 1991, “Touchdown PCR to circumvent spurious priming during gene amplification”, Nucleic Acids Res., 19, 4008; Rodriguez I. R., Mazuruk K. S., Schoen T. J., Chadler J. G., 1994, “Structural analysis of the human hydroxindole-O-methyl transferase gene: presense of two district promoters”, J. Biol. Chem., 269, 31969–31977; Birch D. E., Kolmodin L., W. J., McKinney N., Wong J., Young K. K. K., Zangenberg G. A., Zoccoli M. A., “Simplified hot start PCR”, Nature 381, 445–446, 1996]. Among such efforts to date in the prior art, and for the sack of clarity, standard PCR is described in the present invention. Currently, the most common methodology for PCR involves sample preparation having a master mix and primers [Mullis K. B; Faloona F. A; Scharf S; Saiki R. K; Horn G; Erlich H. A., Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symposia on Quantitative Biology, 1986; and Scharf S. J; Horn G. T; Erlich H. A. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science, Sep. 5, 1986, 233(4768):1076–8.], followed by detection and analysis of the reaction products. Real-time quantitative monitoring of PCR kinetics have been reported [Higuchi R; Fockler C; Dollinger G; Watson R. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology NY, September, 11(9), 1026–30 (1993)] but one important drawback is the requirement for target-specific fluorogenic probes. Several labeling strategies have been reported into literature including radiolabeling, enzyme-linked colorimetry [Yu, H., et al., Cyanine Dye dUTP Analogs for Enzymatic Labeling of DNA Probes, Nucleic Acids Research, 22 (15), pp. 3226–3232, 1994], silver staining, fluorescent staining, and chemoluminescent staining [Zhu, Z., et al., Directly labeled DNA probes using fluorescent nucleotides with different length linkers, Nucleic Acids Research, 22 (16), pp. 3418–3422, 1994].
Higuchi, et al. [1993] described an assay for any amplifiable DNA sequence that uses a video camera to monitor multiple polymerase chain reactions (PCRs) simultaneously over the course of thermocycling. The video camera detects the accumulation of double-stranded DNA (dsDNA) in each PCR using the increase in the fluorescence of ethidium bromide (EtBr) that results from its binding with duplex DNA. The kinetics of fluorescence accumulation during thermocycling are directly related to the starting number of DNA copies. The fewer cycles necessary to produce a detectable fluorescence, the greater the number of target sequences. Results obtained with this approach indicated that a kinetic approach to PCR analysis can quantitate DNA sensitively, selectively, and over a large dynamic range. A commercially available system was developed for real-time PCR assay exclusively exploiting the fluorescence measurement of a pair of reporter-quencher fluorescent probes during thermal cycling reaction [U.S. Pat. Nos. 5,723,591 and 5,210,015].
The basic stages of PCR typically comprise: (1) synthesis of primers complementary to a target piece of DNA, (2) separating strands of DNA target by thermal cycling and attaching primers to each end of the target sequence, (3) extending strands by adding ATP and the enzyme polymerase and then repeating the above steps typically after 25–30 cycles until the replication produces a useful amount of target DNA (e.g., 108 copies). The widespread way to check for the presence of these fragments of DNA of defined length is to load a sample taken from the reaction product, along with appropriate molecular-weight markers, onto an agarose gel which typically contains 0.8–4% Et Br.
DNA bands are then visualized under UV illumination and identification of any product can be done by comparing product bands with reference bands of known molecular-weight markers [Sambrook, et al., Handbook Mol. Biol., Spring Harbor; Gelfand, D. H. and White, T. J., In PCR Protocols, A Guide to Methods & Applications, ed. M. A. Innis, D. H. Gelfand, J. J. Sninsky, T. J. White, 129–41, New-York Academic Press, 1990]. More recently, developments have been reported into the prior art and in particular the introduction of automated DNA synthesizer and new synthesis reagents exploiting multiple fluorophore markers for which changes in fluorescent signal can be proportional to the number of amplification cycles [Handbook of Fluorescent Probes and Research Chemicals, 1996, 6th edition, Molecular Probes Inc. Eugene Oreg.; PCR Systems, Reagents and Consumables, Perkin-Elmer catalog, 1996–1997].
Atwood discloses determination of concentration growth of nucleic acids in PCR using a group of concurrent reaction vessels monitored concurrently by a charge coupled device (CCD) camera [U.S. Pat. No. 5,766,889 to Atwood, John G.]. Atwood disclosures a label-based fluorescence technique.
PCR is the basis of many of today's biotechnology advances. Any improvement in the PCR process has an enormous impact on our lives through biotechnological advances. A MSA combined with a multivariate process would drastically improve PCR performance. Improvements can include better time control, better selectivity, higher purity and lower error rates by not exclusively using tags, and lower cost.
References listed herein are incorporated by reference.